The majority of mitochondrial proteins are encoded by nuclear genes, synthesized in the cytoplasm and targeted directly to the organelle. Two factors contribute to the specificity of protein import into the mitochondria. First the presequence which labels the protein for delivery to the organelle, and second, the components of the cellular machinery that recognize the presequence and facilitate transport of the protein into the organelle. Research is in progress in several laboratories to elucidate the structural requirements of the presequences necessary for mitochondrial localization. The research in this proposal is designed to identify and characterize cellular components which direct mitochondrial protein import by the analysis of yeast (Saccharomyes cerevisiae) chromosomal mutants defective in mitochondrial biogenesis. This analysis will be composed of three parts. a) Selection and preliminary characterization of mutants defective in mitochondrial protein targeting. Gene fusions between the yeast ATP2 gene (B-subunit F1-ATPase) and the E. coli lacZ gene have been shown to encode proteins which cause a respiration-defective phenotype when expressed in yeast. Gly+ revertants of this phenotype include mutants with decreased targeting of the hybrid protein to the mitochondrion. Some of these mutants are also temperature-sensitive and they have been assigned to three complementation groups (mtf-mitochondrial function). Temperature-resistant revertants of mtf mutants include strains (mis-mitochondrial synthesis) that have very low levels of wild-type B-subunit within the mitochondria and are unable to grow on a non-fermentable carbon source. b) Systematic screening of these mtf and mis mutants through a series of assays of mitochondrial structure and function both in vivo and in vitro will provide insight into any defects in mitochondrial biogenesis and identify the most interesting mutants for further analysis. c) In depth analysis of a few of the most interesting mutants to determine the specific role of individual proteins in the import process.